Sustainability in grape production and commerce d Presented by - - PowerPoint PPT Presentation

Sustainability in grape production d and commerce

• Presented by Vincenzo De Luca

Presented by Vincenzo De Luca

– Supported by OMAFRA, NSERC and Industry

• Brent Wiens, MSc
• Dr. Kyung Hee Kim
• Dawn Hall, PhD

Introduction

1. Research Interests: cell specialization and biochemical d ti production 2. Acyltransferases involved in grape volatile production.

• Acyl esters of phenols and terpenes.
• Relationship between anthocyanin 5 glucosides and methyl

anthranilate anthranilate.

3. Glucosyltransferases and grape natural products.

• Water solubility, stability, transport and compartmentation
• Bifunctional GT and its role in resveratrol and phenol tartrates

production production

4. Identification of Grape Pomace secondary metabolites.

• Value as food additives

Sustainability Sustainability

• Wine grapes

– Are clonally propagated L b f id ti l

• Regulatory issues

– Demands by governments to decrease the pesticide – Large numbers of identical individuals growing together – Susceptible to similar diseases U i t i p footprint for wine grape production.

• Contamination of land, water

and air. P l ti i k

– Unique to wine grapes – Lack of large scale breeding efforts

• Unique to wine grapes
• Population risks

– Needs for biocontrol measures

• ver pesticide use

– Need for genetically superior di i t t t – Lack of large scale breeding efforts – Heavy dependence on management and chemical disease resistant genotypes

• Risks

– Appearance of new diseases?

• Pierce’s Disease

g pesticide use

• Powdery Mildew
• New forms of Phylloxera

Approaches to grape improvement Approaches to grape improvement

• Tools available

– Genomics

• Grape genome

sequence (Pinot Noir)

• Examples of important

wine related pathways

Anthocyanins

sequence (Pinot Noir)

• Synteny
• Marker assisted

breeding

– Anthocyanins

• Very well known in plants

– Resveratrol

breeding

– Metabolic profiles – Pathways

G

• Very well known in grape

– Acylated aroma and flavor cpds

• Genes
• Proteins
• Mutations

p

• Methyl anthranilate

– C-13 Norisoprenoids

฀ -damascenone ฀ -damascenone

Cell specific specialization for secondary metabolism

R HO O O CH COOH HC OH COOHGluO

OGlu

Tryptophan Geraniol GH DS

R Phenol tartrates Resveratrol Glucosides OGl u

OCH3 O

TDC Tryptamine Secologanin Strictosidine STR1 DS SLS

OCH3 NH

2

Methyl Anthranilate

Ajmalicine Tabersonine Desacetoxyvindoline Catharanthine D4H DAT T16H SG OH Desacetylvindoline Vindoline DAT N N H H CO C N C2H5 N N HO H CO2CH3 OAc H3CO H3C H H3CO2C

Catharanthus roseus

Anhydrovinblastine Antineoplastic agent

Vincenzo De Luca, Brock University, St. Catharines,Ontario, Canada, February 22, 2010

Catharanthus roseus

Cell factories & biosynthesis of plant natural products

Scanning electron microscopy of aromatic Sage: The Scientist Nov 17 2003 V17: p12

Brock, April 19, 2010

Research Interests of our Laboratory

• Biosynthesis of Novel and commercially important metabolites
• Evolution of biosynthetic pathways
• Cell and biochemical specialization involved

Only 25 h required to fill this gland with

7

fill this gland with monoterpenes and flavonoids

1 2 3 4 5 6

Mint gland on the surface of a mint leaf

8 9 10

mint leaf

CO2

Diagram from: Plant Physiology 136:4215-4227 (2004) photosynthesis

Sugars

2

+ light

Anthocyanin Biosynthesis is the best know pathway of Secondary metabolism in plants

OH 3 malonylCoA + p-coumarylCoA OH O OH HO OH CHS OH OH O O OH HO OH OH O O OH HO CHI F3H OH O O OH HO OH OH OH F3'H F3'5'H OH O O OH HO OH OH O O OH HO OH OH OH O O OH HO OH OH OH OH O O OH HO OH OH OH OH O O OH HO OH OH F3'H F3'5'H FLS FLS DFR DFR OH O O OH HO OH OH O HO OR1 + OH O O OR1 + FLS DFR DFR ANR LDOX ANR LDOX UFGT UFGT O O OR2 HO OGlu O O OR2 HO OGlu OR3

The pattern of grape pigmentation in nine cultivars is regulated by differential gene expression of is regulated by differential gene expression of anthocyanin biosynthesis and their transcription factors

Castellarin and Di Gaspero BMC Plant Biology (2007) 7: 46

Anthocyanin Biosynthesis is the best know pathway of Secondary metabolism in plants

OH 3 malonylCoA + p-coumarylCoA OH O OH HO OH CHS OH OH O O OH HO OH OH OH O O OH HO CHI F3H OH O O OH HO OH OH F3'H F3'5'H F3H OH O O OH HO OH OH O O OH HO OH OH OH O O OH HO OH OH OH OH O O OH HO OH OH OH OH O O OH HO OH OH F3'H F3'5'H FLS FLS OH O O OH HO OH OH O HO OR1 + OH OR1 + FLS DFR DFR ANR LDOX ANR LDOX UFGT UFGT O O OR2 HO OGlu O O OR2 HO OGlu OR3

• The sugar pattern is used to define wines

contaminated with North American grapes contaminated with North American grapes.

• However anthocyanins with sugars on the 5

position are more stable and their presence could be valuable for wine color stability?

Identification of the 5GT responsible for the f ti f th i di l id

OH O HO R1 OH O HO R1

formation of anthocyanin diglucosides

5-GT UDP-Glucose

O O O HO O R2 O HO O OH OH HO HO O O OH HO O R2 O HO OR3 OH OH OH OR3 OH OH

• The inability of most European grapevines to produce

3,5-di-O-glucosides has long been used to classify i di h i i l i i wines according to their varietal origin.

–

This study showed that V. vinifera has a 5GT gene with 2 mutations that render the protein inactive.

–

Correction of the 2 mutations reactivated the V. vinifera 5GT gene

Janvary et al, Agric Food Chemistry (2009) 57: 3512-3518

–

This explains why revertants have not been observed

Colocalization of 5GT and AMAT to the same area of chromosome 9 explains why FOXY & diglucosides are linked!

5-GT

OH O HO R1 R2 OH O HO R1 R2

p y g

UDP-Glucose

O O O O OR3 HO OH O OH OH HO HO O OH O O OR3 HO OH OH OH OH OH

5GT double mutant AMAT-like gene (CAO23156)

• V vinifera (Pinot Noir) genome shows that :
• V. vinifera (Pinot Noir) genome shows that :

– CAO23156 is 95% identical on the amino acid level with V. labrusca anthraniloyl-CoA:methanol anthraniloyl transferase (AMAT). – Colocalization of the two genes would explain genetic linkage between

Janvary et al, Agric Food Chemistry (2009) 57: 3512-3518

these 2 traits in hybrid cultivars.

OH

How are floral

HO HO OH

Lutein Cryptoxanthin

norisoprenoids made?

Journal of Experimental Botany Vol 56

HO

Zeaxanthin

O

VvCCDs

Journal of Experimental Botany, Vol. 56,

• No. 420, pp. 2721–2731, October 2005

www.vcbio.science.ru.nl/. ../applets/chloroplast/

O HO

3-hydroxy- -ionone



Oxidases Reductases & Dehydrogenases

• Grapes

OH O

3-oxo- -ionol



O OH HO

3-hydroxy- -ionol



Oxidases, Reductases & Dehydrogenases

Grapes

– Muscat grapes make more norisoprenoids than in Muscat

• f Alexandria grapes than in

those of Shiraz Si l V CCD t

HO

3-hydroxy-7,8- dihydro- -ionone



HO O

3-hydroxy- -



O

– Single VvCCD gene appears to be involved – VvCCD molecular marker can be used for selection purposes in breeding program to enhance

3 hydroxy damascenone

 -damascenone

g p g the norisoprenoid profiles of wine grapes

How are floral volatile acylated flavor compounds made made?

Example: Example: Grapes make methyl anthranilate from pectin derived methanol and Grapes make methyl anthranilate from pectin derived methanol and the CoA ester of anthranilate Plant J the CoA ester of anthranilate Plant J (2005) 44, 606–619

O H O H HO HO O H H H O

OCH3

O H O H HO HO O H H H O OCH3 O H O H HO OH H H H O

OCH3

the CoA ester of anthranilate Plant J the CoA ester of anthranilate Plant J (2005) 44, 606 619

O O H HO O H H O OH O H HO O H H H O OH O OH

Pectin Methylesterase

Cell Wall softening

O H HO H O H HO H O OH O H HO OH O H H H

MeOH

O SCoA O OCH3 O OH

Ce a so e g during ripening

92 4 2 2

+

NH2 NH2 AMAT NH2 Coenzyme A Ligase

% distribution

• f AMAT

Anthraniloyl CoA Methyl anthranilate Anthranilic acid

In grape Cross-section

Concord wk16

European Grape cultivars have almost identical transcripts to those of AMAT of identical transcripts to those of AMAT of Concord Grape?

Different varieties of mature Vitis vinifera grapes contain AMAT-like acyltransferase (pAAT) transcripts. The arrow denotes positives: a 331 base pair product amplified with primers designed against the C-terminal region of the pAAT. Varieties in blue font were selected for cloning of a full length cDNA gene selected for cloning of a full-length cDNA gene.

His tag k putative AMAT-like gene sequence t ki His tag f1 origin T7 terminator

XhoI (1) NcoI (663) NcoI (1350 )

flPAAT in pet30(b)+

67 17 bp

kan sequence T7 promoter enterokinase thrombin S tag His tag lac I lac operator ColE1 pBR322 origin

Grape varieties produce slightly different transcripts responsible for the short truncated proteins produced in Cabernet franc, Chardonnay Musque and Shiraz cultivars

410 420 430 440 450 460 470

( 410)

CCACGACAATTCTCGGTTCCCCTCTTATCTTAATTCAGGTGACCCGATTGAGGTGCGGAGGATTT

AMAT ( 410)

CCACGAGAATTCTTGGTTCCCCTCTTATCTTAATTCAGGTGACCCGATTGAGGTGCGGGGGATTT

Sauvignon Blanc pAAT ( 410)

CCACGAGAATTCTTGGTTCCCCTCT-ATCTTAATTCAGGTGACCCGATTGAGGCGCGGGGGGTTT

Cabernet Franc pAAT ( 410)

CCACGAGAATTCTTGGTTCCCCTCT-ATCTTAATTCAGGTGACCCGATTGAGGCGCGGGGGGTTT

Shiraz pAAT ( 410)

CCACGAGAATTCTTGGTTCCCCTCT-ATCTTAATTCAGGTGACCCGATTGAGGCGCGGGGGGTTT

Chardonnay Musque pAAT ( 410)

Shiraz cultivars.

• Sauvignon blanc grapes

49 kDa make a protein of the appropriate size & a truncated protein as well?

• Other cultivars only

24 kDa Other cultivars only accumulate truncated protein forms.

• Do truncated proteins have
• ther biological activities?

18 kDa

• ther biological activities?
• What is the biochemical role
• f Sauvignon blanc

acyltransferase?

Summary of Vitis vinifera AMAT- Lik i Like protein

• One full-length functionally active form of acyltransferase

g y y exists

• One mutated form that produces a truncated protein

exists exists

• Scan all commercial grape varieties for presence of the

active form during grape ripening.

Documentation of genes expressed vs useful wine metabolites – Documentation of genes expressed vs useful wine metabolites produced – Fingerprint of the genetic make up of the cultivar

Use as a marker for introduction of the trait in other

• Use as a marker for introduction of the trait in other

vinifera cultivars

– Breeding vs genetic engineering

Grape Glucosyltransferases Grape Glucosyltransferases

• Grapes have hundreds of

GT GTs

• Their roles are to

increase the solubility and t bilit f d stability of secondary metabolites such as anthocyanins Glycosides may be

• Glycosides may be

transported or stored in vacuoles

• Glycosides may also
• Glycosides may also

‘activate’ the molecule for further reactions and biosynthesis biosynthesis

Grapes accumulate resveratrol in the form f Gl id

NH3 COOH

• f Glucosides
• Derived from

Phenylalanine

COOH COOH HO phenylalanine PAL C4H

Phenylalanine

• Common pathway to

flavonoids and anthocyanins

HO p-coumaric acid p-coumaroyl CoA Ligase cinnamic acid

anthocyanins

• Unique polyketide

synthase for assembly of

• Glucosylation to

solubilize and accumulate resveratrol within

3 malonylCoA + p-coumarylCoA Resveratrol synthase

assembly of resveratrol plant vacuoles in ripening grape skins

HO OH

GluO OH HO OGlu

OH Resveratrol

OH Trans-piceid OH Trans-resveratroloside

Bifunctional rVLRSgt of Concord grape Bifunctional rVLRSgt of Concord grape

COOH HO H3CO COOH HO HO COOH HO H3CO

HO OH O OH OH HO O O HO HO HO OH

OCH3 HO HO HO Synapic Caffeic Ferulic COOH HO COOH HO COOH HO COOH HO

OH 1) t-Resveratrol OH O OH O OH OH HO OH 2) Kaempferol HO 3) Esculetin O OH HO O OH OH HO OH OH 4) c-Resveratrol O OH OGlu Ho OH H

+

Coumaric p-OH-Benzoic Benzoic Coumaric

OH O OH 5) Quercetin O OH 6) Naringenin O OH 7) Dihydroquercetin OGlu OH 8) Kuromanin

250 500

vity (pkatal/mg)

MES pH 6.0 T i H 9 0

2500 5000

ctivity (pkatal/mg)

MES pH 6.0 Tris pH 9.0

t

• R

e s v e r a t r

• l

K a e m p f e r

• l

E s c u l e t i n c

• R

e s v e r a t r

• l

Q u e r c e t i n N a r i n g e n i n D H Q K u r

• m

a n i n

Specific activ

Tris pH 9.0

Sinapic acid Ferulic acid Caffeic acid Coumaric acid pHBA Benzoic acid Cinnamic acid

Substrates Specific ac t c Substrates

Biotechnological production of substituted resveratrols

3 6 8 9 10 11 12 13 14 15 16

G ll &

12 13 14 15 16

Grape callus & cell suspension cultures Cloning of functional Resveratrol Glucosyltransferase & transfer of functional gene to b t i Pl t J (2006) 49 579 Alanine triggers resveratrol biosynthesis & O H OH

OH

• bacteria. Plant J (2006) 49: 579–

591 biosynthesis & accumulation OH O H

GluO OH

Bacteria expressing Resveratrol GT

Plant Science (2006) 171:

OH

Resveratrol GT

( ) 734–744 Collaboration with Christophe Clement, Eric Courot, David Donnez, Philipe Jeandet

Involvement of VLRSgt in biosynthesis glucose esters and biological role in biosynthesis of caffeoyl esters and biological role in biosynthesis of caffeoyl tartrate

• Caftaric acid

COOGlu COGlu H3CO

– Major phenol in wine – Involved in browning reaction

COOGlu HO HO COOGlu HO H3CO HO HO

reaction – Reacts with glutathione to produce S-glutathionyl caftaric acid

HO OCH3 HO Phenolic Glucose Ester Pool

caftaric acid – Quantities in wine >30mg/l

HO O O CH COOH HC OH COOH HO HO COOH

Caffeoyltartrate

• r

Caftaric acid

CaffeoylGlucose:tartrate caffeoyltransferase

Malic Tartaric acid

OH OH

Grape

NH2 OH OH OGlucose OH OH

Grape

Phenylalanine

OH HO OH HO OH HO OGlucose HO OH HO OTartrate HO

Coumaric acid Coumaroyl glucose Coutaric acid

OH HO HO OH H3CO OGlucose OH H3CO OGlucose HO HO OTartrate OH H3CO OTartrate HO

Caffeic acid Caffeoyl glucose Caftaric acid

OH HO H3CO OH OH OGlucose HO OH OTartrate HO

Ferulic acid Feruloyl glucose Fertaric acid

OH OCH3 HO H3CO OGlucose OCH3 HO H3CO OMalate OH OCH3 HO H3CO

Synapic acid Synapoyl glucose Synapoylmalate

Arabidopsis

Synapic acid Synapoyl glucose Synapoylmalate

Characterization of the Nutraceutical Value of (VFL) G d d (VFL) Grape pommace sourced powder

• Identifying antioxidant

Free and Bound

y g activity and other health benefits –Polyphenols in this powder

Free and Bound secondary metabolites.

in this powder

– Flavonoids – Anthocyanins – Proanthocyanidins – Catechins – Triterpenes (Saponins) p ( p )

UPLC MS facility for metabolite analysis

– Screening capabilities based on UPLC t f W t system from Waters.

• Traditional HPLC Systems normally take 30

to 45 min to run a sample (32 to 48/24 hr)

• UPLC has been standardized to run 7 min

per sample (205/24hr)

• If a particular peak is desired, the run can

be decreased to 1min (1440/24 hr)

• Very short run times suggest system can be

y gg y used for mutant screens

• Program can be produced to automatically

select promising candidates

UPLC chromatogram of SOLUBLE phenolic acids in pomace powder, wet pomace and seed ac ds po ace po de , et po ace a d seed pomace at 280 nm

1) gallic acid 2) protocatechuic acid 3) Catechin 4) procyanidin B 5) procyanidin B 6) Epicatechin 6) Epicatechin 7) quercetin glucuronide 8) quercetin.

UPLC chromatogram of BOUND phenolic acids in pomace powder, wet pomace and seed pomace at 280 p p , p p nm

P1 gallic acid P2 protocatechuic acid P2 protocatechuic acid P3 ρ-hydroxybenzoic acid P4 gentisic acid P5 caffeic acid P6 (-)-epicatechin 6 ( ) ep catec P7 ρ-coumaric acid

Table 1. Concentration of major phenolic acids in grape pomaces (μg/g) Methanol extract Residue Phenolic Powder Wet pomace Seed Powder Wet pomace Seed acids Powder Wet pomace Seed Powder Wet pomace Seed Gallic acid 260 12 511 3398 990 874 Catechin 111 23 237

• Epicatechin

280 412 166 1132 856 232 Epicatechin 280 412 166 1132 856 232 Caffeic acid

• 111

80 31 * ( ) % * ( ) has been calculated as a percentage, %.

• Anthocyanins in wet pomace

and pomace powder and pomace powder

– P1) dephinidin – P2) cyanidin P3) l idi – P3) malvidin

Table: [major anthocyanins] in grape pomaces (μg/g) Table: [major anthocyanins] in grape pomaces (μg/g) Delphinidin Cyanidin Malvidin

3 1 2 388 Wet pomace 35 172 388 Pomace powder 32 117 165

* ( ) has been calculated as a percentage, %.

Triterpenes in grape pomace power

(D) Dry pomace (W) W t (W) Wet pomace (S) grape seed (SK) fresh grape skin (SC) seed coat ( )

Summary of grape pomace metabolites id tifi d b UPLC ESI MS identified by UPLC-ESI-MS.

OH

• Analyses of different

grape pomaces:

Little or No res eratrol

HO OH

– Little or No resveratrol found in wet or dry pomace.

OH Resveratrol OH OH

– We have identified pomace source with significant levels of

HO O HO

significant levels of resveratrol & viniferin can be found

HO OH Viniferin

Summary of grape pomace metabolites id tifi d b UPLC ESI MS identified by UPLC-ESI-MS.

COOH

• Main Phenolic acid: Gallic

HO OH OH COOH

acid

• Procyanidin (+-catechin)
• dephinidin cyanidin and

HO OH OH Gallic acid

• dephinidin, cyanidin and

malvidin as major anthocyanins. W t t i d

OH OH (+)-catechin OH OH

• Wet pomace contained

higher concentration of anthocyanins than d d d

O OH OH HO OH Delphinidin

+

CH3 CH3 COOH CH3 H3C

powder and seed pomace

• High content of oleanolic

acid

Delphinidin

HO CH3 CH3 H3C

Oleanolic acid

Summary: Future Prospects Summary: Future Prospects

• The biochemistry and molecular biology of wine chemistry is rapidly being

characterized characterized

– Anthocyanins – Resveratrol – AMAT-like aroma compounds – C-13 norisoprenoids Ph li – Phenolics – Terpenes

• Tools will be used to:

– Fingerprint differences between cultivated wine grapes

• Marketing and commecial protection of cultivars

Marketing and commecial protection of cultivars

• Identity preservation

– So far documented differences appear to be small involving single or double gene mutations – Breeding programs (Classical genetics/Genetic Engineering)to improve

• Color, Flavor and Aroma
• Stress tolerance of grapes (Cold, Drought, Disease, Herbivore)

Stress tolerance of grapes (Cold, Drought, Disease, Herbivore)

– Grape Pomace analysis and potential uses as value added sources of anti-oxidants

• Improved marketing of byproducts for incorporation into foods
• New health claims
• Identity preservation of certain pomace sources